CN112940716A - Fluorescence method for rapidly detecting bacteria based on fluorescent probe - Google Patents

Fluorescence method for rapidly detecting bacteria based on fluorescent probe Download PDF

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CN112940716A
CN112940716A CN201911258659.6A CN201911258659A CN112940716A CN 112940716 A CN112940716 A CN 112940716A CN 201911258659 A CN201911258659 A CN 201911258659A CN 112940716 A CN112940716 A CN 112940716A
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bacteria
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pyrene
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徐兆超
刘卫卫
苗露
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Dalian Institute of Chemical Physics of CAS
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Abstract

The invention provides a fluorescence method for rapid bacteria detection, which uses a ratiometric fluorescent probe with the structure shown in (1), and emits pyrene monomer fluorescence at 375nm and pyrene exciplex fluorescence at 482 nm. The compound forms nano aggregates due to hydrophobic effect in aqueous solution to show weak exciplex fluorescence, after bacteria are added, the probe is positively charged, the surface of the bacteria is greatly negatively charged, and the aggregates are disaggregated due to the action of the probe and the bacterial cell wall to show fluorescence enhancement signals. Because the surface of different bacteria has slight difference, the difference can be caused in the fluorescence enhancement degree, and different bacteria types can be distinguished by using the fluorescence method.

Description

Fluorescence method for rapidly detecting bacteria based on fluorescent probe
Technical Field
The invention belongs to the field of biological analysis and detection, and particularly relates to a fluorescence method for rapidly detecting bacteria.
Background
The bacteria exist in the aspect of life, most of the bacteria are beneficial bacteria and have no harm to human, and the existence of part of pathogenic bacteria is directly related to the development of diseases, thus seriously threatening the health of human. When these pathogenic bacteria are present in a growth-promoting environment, they can multiply and multiply in a short time and cause cross-infection among patients, which is one of the major causes of serious complications and death of patients in hospitals. The rapid and sensitive bacteria detection method can guide doctors to reasonably treat, thereby controlling the spread and cross infection of bacteria, improving the survival rate of patients with bacterial infection and effectively preventing epidemic diseases.
Currently, the routine detection methods for bacteria in hospitals are bacterial blood culture and phenotypic identification of microorganisms, which are the current clinical gold standard. However, this method is cumbersome and the entire identification process is very time-consuming (days or even weeks) and often relies on the expertise of the operator. Alternatively, certain genotyping methods, such as real-time Polymerase Chain Reaction (PCR) and Fluorescence In Situ Hybridization (FISH), can effectively reduce assay time to hours (about 6-10 hours) after being positive for blood culture. However, their widespread use is still hampered by the limited detection sensitivity (e.g., <100CFU mL "1) and the lack of specificity and high background signal generated by the surrounding large number of non-target species (e.g., red blood cells). Furthermore, all of these conventional methods involve complicated and laborious sample handling (e.g., cell culture, cell lysis, nucleic acid extraction, and PCR amplification) to some extent, which makes high-throughput analysis of clinical samples difficult. Therefore, there is an urgent need to develop rapid, multiplex and ultrasensitive methods for high-throughput identification of pathogenic bacteria in clinical diagnosis.
The fluorescence probe method has the characteristics of rapidness and sensitivity, and is widely developed in recent years for rapidly detecting bacteria. For example, the fluorescence sensor array method is reported to be used for distinguishing and detecting bacteria, since various bacteria have different surface characteristics (mainly charge characteristics), non-specific interaction between different fluorescence probes in the sensor array and different bacteria surfaces generates different mode signals, and finally the fluorescence sensor array identifies the bacteria by reading the different mode signals. But the types of bacteria that can be diagnosed by the fluorescence sensor array are limited due to insufficient selectivity and sensitivity.
Pyrene, a classical fluorophore, gives different response signals depending on the mode of action. We designed a simple sensor molecule with pyrene fluorophore as the signal molecule and developed a simple fluorescence method for rapid identification of different species of bacteria with this as the core.
Disclosure of Invention
The object of the present invention is to provide a fluorescent method for rapid bacterial detection. The ratiometric fluorescent probe used in the method forms nano-aggregates due to hydrophobic effect in aqueous solution to show weaker exciplex fluorescence, after bacteria are added, the probe is positively charged, the surface of the bacteria is greatly negatively charged, and the aggregates are disaggregated due to the action of the probe and the bacterial cell wall to show fluorescence enhancement signals. Because the surface of different bacteria has slight difference, the difference can be caused in the fluorescence enhancement degree, and different bacteria types can be distinguished by using the fluorescence change. The method has the characteristics of simple preparation method and sensitive and quick detection, and the performance of the method is superior to that of the existing bacteria detection probe.
The invention provides a fluorescent probe which can emit pyrene monomer fluorescence at 375nm and pyrene exciplex fluorescence at 482 nm. The structural formula is as follows:
Figure BDA0002311003010000021
the compound forms nano aggregates due to hydrophobic effect in aqueous solution to show weak exciplex fluorescence, after bacteria are added, the probe is positively charged, the surface of the bacteria is greatly negatively charged, and the aggregates are disaggregated due to the action of the probe and the bacterial cell wall to show fluorescence enhancement signals. Because the surface of different bacteria has slight difference, the difference can be caused in the fluorescence enhancement degree, and different bacteria types can be distinguished by using the fluorescence method.
A synthetic method and a synthetic route of a ratiometric fluorescent probe used in a fluorescence method for rapidly detecting bacteria are as follows:
Figure BDA0002311003010000031
the method comprises the following specific steps:
(1) synthesizing an intermediate 3-bromo-1-pyrenyl acetone:
under the protection of nitrogen, dissolving pyrene and 3-bromopropionyl bromide in dichloromethane, cooling to-5-0 ℃ in an ice salt bath, slowly adding anhydrous aluminum chloride into the reaction liquid under stirring, and stirring for 1-3 hours to obtain the reaction liquid with a dark color; heating the reaction temperature to room temperature, continuously stirring for 6-10 hours, pouring the reaction liquid into ice water, extracting with dichloromethane, drying with anhydrous magnesium sulfate, filtering, removing the solvent by spinning, and separating by silica gel column chromatography to obtain 3-bromo-1-pyrenyl acetone;
(2) synthesis of intermediate 1- (3-bromopropyl) -pyrene:
under the protection of nitrogen, dissolving anhydrous aluminum chloride and lithium aluminum hydride in diethyl ether, dissolving 3-bromo-1-pyrenyl acetone in dichloromethane, slowly adding the dichloromethane into the reaction solution, stirring at room temperature for reaction for 0.5-3 hours, pouring the reaction solution into ice water, quenching the rest of aluminum chloride and lithium aluminum hydride, adjusting the pH of the reaction solution to acidity by hydrochloric acid, then extracting by dichloromethane, drying by anhydrous magnesium sulfate, filtering, removing the solvent by spinning, and performing silica gel column chromatographic separation to obtain 1- (3-bromopropyl) -pyrene;
(3) synthesis of ratiometric fluorescent Probe PI-3 in response to Membrane Charge:
under the protection of nitrogen, dissolving biimidazole and 1- (3-chloropropyl) -pyrene in acetonitrile, heating to 80-140 ℃, reacting for 24-96 hours under stirring, stopping the reaction, separating out white precipitate, filtering, and washing the precipitate with diethyl ether; the obtained precipitate was then dissolved in methanol, to which a saturated methanol solution of potassium hexafluorophosphate was slowly added dropwise to precipitate a white precipitate product, i.e., ratiometric fluorescent probe PI-3, which responded to membrane charge.
In the step (1), pyrene: the mass ratio of the 3-bromopropionyl bromide is 1: 0.3-2;
pyrene: the mass ratio of the anhydrous aluminum chloride is 1: 0.3-3;
the volume ratio of the mass of pyrene to the volume of dichloromethane is 1:5-50 g/mL.
In the step (2), the intermediate 3-bromo-1-pyrenyl acetone: the mass ratio of the lithium aluminum hydride is 1: 0.2-1;
intermediate 3-bromo-1-pyrenyl acetone: the mass ratio of the anhydrous aluminum chloride is 1: 0.5-2;
the volume ratio of the mass of the intermediate 3-bromo-1-pyrenyl acetone to the diethyl ether is 1:3-15 g/mL.
The volume ratio of the mass of the intermediate 3-bromo-1-pyrenyl acetone to the dichloromethane is 1:15-90 g/mL.
In the step (3), the intermediate 1- (3-bromopropyl) -pyrene: the mass ratio of the bigeminal imidazole is 15-4: 1;
the volume ratio of the mass of the intermediate 1- (3-bromopropyl) -pyrene to the acetonitrile is 1:20-200 g/mL.
A fluorescence method for rapidly detecting bacteria comprises the following specific detection methods:
(1) shaking and culturing the bacteria at 37 ℃ by a shaking table;
(2) the bacteria were centrifuged at 13000rpm for 2 minutes, the supernatant was discarded, the bacteria were washed 2-3 times with 20mM PBS buffer at pH 7.4, and finally the bacteria were resuspended in PBS buffer;
(3) the bacteria were probed with a fluorescence spectrometer.
Culturing the bacteria to OD in the step (1)600＝0.1-2.0。
Resuspending to OD of bacteria with PBS in step (2)6000.1-2.0, final concentration of probe 5-20 μ M.
In step (3), fluorescence is detected at an excitation wavelength of 345nm to obtain a fluorescence spectrogram, and the change in fluorescence ratio (I)482/I375) On the abscissa, the fluorescence increases (Δ S/S)0) Two-dimensional plots were generated for the ordinate, to distinguish between different species of bacteria.
The invention has the advantages and beneficial effects that:
the interaction between the probe with positive charge and the bacteria with a large amount of negative charge on the surface causes the fluorescence to be greatly enhanced. Due to different surface charge distributions of different kinds of bacteria, the probes form monomers and dimers to different degrees, so that the ratio change of pyrene monomer and exciplex fluorescence is generated (I)482/I375). The fluorescence is obtained by taking the difference of fluorescence enhancement generated after the probe polymer is combined with different bacteria and the change of the ratio of the monomer to the exciplex fluorescenceSignal, capable of distinguishing between different species of bacteria.
Drawings
FIG. 1 shows the hydrogen nuclear magnetic spectrum of the fluorescent probe prepared in example 1;
FIG. 2 is a carbon spectrum of nuclear magnetic spectrum of the fluorescent probe prepared in example 1;
FIG. 3 shows the DMSO/H ratio of the fluorescent probe prepared in example 1 and described in example 22Ultraviolet-visible absorption spectra in O mixed liquor;
FIG. 4 shows the DMSO/H ratio of the fluorescent probe prepared in example 1 and described in example 22Fluorescence emission spectrum of O mixed solution;
FIG. 5 is a dynamic light scattering spectrum of the fluorescent probe prepared in example 1 in PBS buffer (20mM, pH 7.4) as described in example 4
Fig. 6 is a scanning electron micrograph of the fluorescent probe prepared in example 1 in PBS buffer (20mM, pH 7.4) as described in example 5.
FIG. 7 is a graph showing fluorescence spectra of the fluorescent probe prepared in example 1 before and after the reaction with different concentrations of Bacillus subtilis in PBS buffer (20mM, pH 7.4) as described in example 6.
FIG. 8 is a graph showing fluorescence spectra of the fluorescent probe prepared in example 1 before and after the reaction with different concentrations of E.coli in PBS buffer (20mM, pH 7.4) as described in example 7.
FIG. 9 shows the fluorescent probe prepared in example 1 and described in example 8, in PBS buffer (20mM, pH 7.4) against different bacteria (OD)6000.5) fluorescence spectra before and after the action and two-dimensional ratio plots.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1
The preparation of ratiometric fluorescent probes for use in the fluorescence method for rapid detection of bacteria, the basic synthesis method is as follows:
(1) synthesizing an intermediate 3-bromo-1-pyrenyl acetone:
pyrene (4g,19.77mmol) and 3-chloropropionyl bromide (2.83g, 16).48mmol) was dissolved in 40mL of anhydrous dichloromethane. The reaction temperature was reduced to 0 ℃ under nitrogen protection. Adding AlCl3Powder (2.64g,19.77mmol) was added to the reaction mixture and stirred until the reaction mixture turned dark red, and the temperature was slowly raised to room temperature for overnight reaction. Adding ice water to quench the unreacted AlCl in the reaction solution3The product is extracted by adding dichloromethane, the organic phase is washed with saturated NaCl solution and MgSO is added4Dry overnight. Dichloromethane was distilled off under reduced pressure, and the residue was separated by silica gel column separation (diethyl ether: dichloromethane ═ 7: 3, V/V) to give 3-chloro-1-pyrenylacetone.
(2) Synthesis of intermediate 1- (3-bromopropyl) -pyrene:
LiAlH is added under the protection of nitrogen4(0.295g,7.78mmol) and AlCl3(1.04g,7.78mmol) was dissolved in 9mL of anhydrous ether. The compound 3-chloro-1-pyrenylacetone (0.910g,3.11mmol) dissolved in 12.5mL of dichloromethane was slowly added to the reaction solution and stirred at room temperature for 1 h. Adding ice water to quench the unreacted AlCl in the reaction solution3And LiAlH4The product is extracted by adding dichloromethane, the organic phase is washed with saturated NaCl solution and MgSO is added4Dry overnight. The methylene chloride was distilled off under reduced pressure, and the residue was subjected to silica gel column separation (petroleum ether) to obtain 1- (3-chloropropyl) -pyrene.
(3) Synthesis of Probe PI-3:
under the protection of nitrogen, the compounds 1- (3-chloropropyl) -pyrene (0.439g,1.58mmol) and diimidazomethane (0.10g,0.7mmol) were dissolved in 20mL acetonitrile and refluxed for 24h to obtain a precipitate. The precipitate was filtered off and washed with diethyl ether. Dissolving the precipitate in 25mL of methanol solution, and slowly dropping saturated KPF into the solution6The solution was filtered and dried to obtain PI-3 as shown in FIGS. 1 and 2.
The hydrogen and carbon spectra data for the PI-3 probe molecules are as follows:
1H NMR(400MHz,DMSO-d6)δ9.81(s,2H),8.48-7.71(m,20H),6.78(s,2H),4.42(t,J＝7.2Hz,4H),3.47-3.29(m,4H),2.34(dt,J＝15.2,7.6Hz,4H).
13C NMR(100MHz,DMSO-d6)δ138.11,135.40,131.33,130.82,130.02,128.57,127.92,127.86,127.74,127.24,126.74,125.63,125.46,125.41,124.71,124.52,123.71,123.70,122.75,58.90,49.72,31.69,29.66.
example 2
The preparation of fluorescent probe for fast bacteria identification includes the following steps:
(1) synthesizing an intermediate 3-bromo-1-pyrenyl acetone:
under the protection of nitrogen, sequentially adding 2.0g of pyrene, 0.6g of 3-bromopropionyl bromide and 10mL of dichloromethane into a 100mL double-mouth bottle, cooling to-10 ℃ by using an ice salt bath, slowly adding 0.6g of anhydrous aluminum chloride into the reaction liquid under the stirring condition, and continuously stirring for 1 hour to obtain the reaction liquid with a dark color; removing the ice bath, slowly raising the reaction temperature to room temperature, continuing stirring for 10 hours, stopping the reaction, pouring the reaction liquid into 100mL of ice water, then extracting with dichloromethane for three times, drying with anhydrous magnesium sulfate, filtering, removing the solvent by rotation, and separating by a silica gel column to obtain a light yellow solid, namely 3-bromo-1-pyrenyl acetone with the yield of 30%.
(2) Synthesis of intermediate 1- (3-bromopropyl) -pyrene:
under the protection of nitrogen, 1g of anhydrous aluminum chloride, 0.6g of lithium aluminum hydride and 6mL of ether are sequentially added into a 250mL Schlenk bottle, 2g of 3-bromo-1-pyrenyl acetone is dissolved in 30mL of dichloromethane under the stirring condition, the mixture is slowly added into the reaction liquid, the mixture is stirred and reacted for 3 hours at room temperature, the reaction liquid is poured into ice water, the rest of aluminum chloride and lithium aluminum hydride are quenched, the pH value of the reaction liquid is adjusted to be acidic by hydrochloric acid, then the reaction liquid is extracted by dichloromethane, dried by anhydrous magnesium sulfate, filtered, the solvent is removed by rotation, and silica gel column chromatography is carried out to obtain white solid 1- (3-bromopropyl) -pyrene with the yield of 33%.
(3) Synthesis of Probe PI-3:
under the protection of nitrogen, sequentially adding 10mg of biimidazole, 150mg of 1- (3-bromopropyl) -pyrene and 3mL of acetonitrile into a 50mL single-mouth bottle, heating to 110 ℃, reacting for 96 hours under stirring, stopping the reaction, separating out white precipitate, filtering, and washing the precipitate with diethyl ether; the resulting precipitate was then dissolved in methanol, to which was slowly added dropwise a saturated methanol solution of potassium hexafluorophosphate to precipitate a white precipitate product with a yield of 8%.
The nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:
1H NMR(400MHz,DMSO-d6)δ9.81(s,2H),8.48-7.71(m,20H),6.78(s,2H),4.42(t,J＝7.2Hz,4H),3.47-3.29(m,4H),2.34(dt,J＝15.2,7.6Hz,4H).
13C NMR(100MHz,DMSO-d6)δ138.11,135.40,131.33,130.82,130.02,128.57,127.92,127.86,127.74,127.24,126.74,125.63,125.46,125.41,124.71,124.52,123.71,123.70,122.75,58.90,49.72,31.69,29.66.
the product structure is identified as probe PI-3.
Example 3
The preparation of fluorescent probe for fast bacteria identification includes the following steps:
(1) synthesizing an intermediate 3-bromo-1-pyrenyl acetone:
under the protection of nitrogen, sequentially adding 2.0g of pyrene, 4g of 3-bromopropionyl bromide and 100mL of dichloromethane into a 100mL double-mouth bottle, cooling to-10 ℃ by using an ice salt bath, slowly adding 6g of anhydrous aluminum chloride into the reaction liquid under the stirring condition, and continuously stirring for 1 hour to obtain the reaction liquid with a dark color; removing the ice bath, slowly raising the reaction temperature to room temperature, continuing stirring for 10 hours, stopping the reaction, pouring the reaction liquid into 100mL of ice water, then extracting with dichloromethane for three times, drying with anhydrous magnesium sulfate, filtering, removing the solvent by rotation, and separating by using a silica gel column to obtain a light yellow solid, namely 3-bromo-1-pyrenyl acetone with the yield of 75%.
(2) Synthesis of intermediate 1- (3-bromopropyl) -pyrene:
under the protection of nitrogen, 4g of anhydrous aluminum chloride, 2g of lithium aluminum hydride and 30mL of diethyl ether are sequentially added into a 250mL Schlenk bottle, 2g of 3-bromo-1-pyrenyl acetone is dissolved in 180mL of dichloromethane under the stirring condition, then the mixture is slowly added into the reaction solution, the mixture is stirred and reacted for 3 hours at room temperature, the reaction solution is poured into ice water, the rest of aluminum chloride and lithium aluminum hydride are quenched, the pH value of the reaction solution is adjusted to be acidic by hydrochloric acid, then the reaction solution is extracted by dichloromethane, dried by anhydrous magnesium sulfate, filtered, the solvent is removed by rotation, and silica gel column chromatography is carried out to obtain white solid 1- (3-bromopropyl) -pyrene with the yield of 36%.
(3) Synthesis of Probe PI-3:
under the protection of nitrogen, sequentially adding 25mg of biimidazole, 100mg of 1- (3-bromopropyl) -pyrene and 20mL of acetonitrile into a 50mL single-mouth bottle, heating to 110 ℃, reacting for 96 hours under stirring, stopping the reaction, separating out white precipitate, filtering, and washing the precipitate with diethyl ether; the resulting precipitate was then dissolved in methanol, to which saturated potassium hexafluorophosphate methanol solution was slowly added dropwise, to precipitate a white precipitate product in 11% yield.
The nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:
1H NMR(400MHz,DMSO-d6)δ9.81(s,2H),8.48-7.71(m,20H),6.78(s,2H),4.42(t,J＝7.2Hz,4H),3.47-3.29(m,4H),2.34(dt,J＝15.2,7.6Hz,4H).
13C NMR(100MHz,DMSO-d6)δ138.11,135.40,131.33,130.82,130.02,128.57,127.92,127.86,127.74,127.24,126.74,125.63,125.46,125.41,124.71,124.52,123.71,123.70,122.75,58.90,49.72,31.69,29.66.
the product structure is identified as probe PI-3.
Example 4
Examination of aggregation Properties of fluorescent probes prepared in example 1
Dissolving the probe in DMSO to prepare 2mM mother liquor, and dissolving the probe in DMSO/H with water content of 0%, 20%, 40%, 60%, 80%, 99.5% respectively2In the O mixed solution, the probe was added to a final concentration of 10. mu.M, and then the UV-visible absorption spectrum was measured to obtain FIG. 3.
In pure DMSO solution in FIG. 3, the probe has strong UV-visible absorption peaks at 256nm, 278nm, 324nm, and 345nm with H2With the addition of O, the intensity of the UV-visible absorption peak of the probe decreased and a slight blue shift appeared indicating a gradual aggregation of the probe.
Example 5
Examination of aggregation Properties of fluorescent probes prepared in example 1
Dissolving the probe in DMSO to prepare 2mM mother liquor, and dissolving the probe in DMSO/H with water content of 0%, 20%, 40%, 60%, 80%, 99.5% respectively2In the O mixture, the final concentration of the probe was 10. mu.M, and then the concentration was adjusted to 345nm at the excitation wavelengthThe fluorescence spectrum gave the result of FIG. 4.
In FIG. 4, the probe shows a strong peak of pyrene monomer at 375nm and an excimer peak of pyrene at 482nm in a pure DMSO solution under excitation of excitation light at 345 nm. With H2When O is added, the intensity of the fluorescence peak of the probe pyrene monomer is gradually reduced, which indicates that the probe is aggregated in the aqueous solution.
Example 6
Size study of aggregates formed by the fluorescent probes prepared in example 1
The probe was dissolved in DMSO to prepare a 2mM stock solution, and then prepared into 5 μ M, 10 μ M, and 20 μ M solutions in 1mL of a 20mM PBS solution at pH 7.4, and the particle size was measured by a dynamic light scattering instrument to obtain fig. 5.
In FIG. 5, the average size of the particles formed with only 5. mu.M PI-3 probe was 366.2nm, the average size of the particles formed with 10. mu.M probe was 593.8nm, and the average size of the particles formed with 20. mu.M probe was 805.7nm, and it can be seen that the size of the particles formed by probe aggregation also increased with increasing probe concentration.
Example 7
Example 1 investigation of surface morphology of the prepared fluorescent Probe
The probe was dissolved in DMSO to prepare a 2mM stock solution, and then prepared into a 10 μ M solution in 1mL of a 20mM PBS solution at pH 7.4, and the morphology thereof was observed by a scanning electron microscope to obtain fig. 6.
In FIG. 6 only PI-3 probe was present in PBS (10. mu.M) to form first small nanoparticles, which were then agglomerated together to form large clusters of particles.
Example 8
Example 1 study of the fluorescence spectra of the action of the prepared fluorescent probe with different concentrations of Bacillus subtilis
Taking 100 mu L of bacillus subtilis to shake culture in 20mL of liquid culture medium at 37 ℃ in a shaking table until the bacillus subtilis grows to the bacterial concentration OD6001.0-2.0, dispensing into small EP tubes, trimming, centrifuging at 13000rpm for 2 min, discarding the supernatant, and diluting with 20mM pH 74 washing the bacteria 2-3 times with PBS buffer, finally resuspending the bacteria with PBS buffer, and then preparing the OD600Bacterial solutions with values of 5, 2, 1, 0.5, 0.1, 0.05, 0.02.
Dissolving the probe in DMSO to prepare 2mM mother liquor, adding 1mL of bacillus subtilis liquid with different concentrations and 20mM of PBS solution with pH of 7.4 into 5 muL of probe respectively, blowing and beating uniformly, wherein the final concentration of the probe is 10 muM, and detecting fluorescence under 345nm excitation wavelength to obtain a fluorescence spectrogram 7.
In FIG. 7, the peaks at 375nm and 482nm were very low in the presence of only the probe, and the peaks at 375nm and 482nm were significantly increased after the addition of Bacillus subtilis. The peak at 375nm gradually increased with increasing concentration of Bacillus subtilis; while the peak at 482nm tends to increase first and then decrease. In particular, the concentration of the Bacillus subtilis is determined from OD6000.02 to OD600The peak at 482nm increased with increasing bacillus subtilis concentration from OD 6001 to OD600The peak at 482nm decreases with increasing bacillus subtilis concentration, 10.
Example 9
Example 1 investigation of the fluorescence spectra of the fluorescent probes prepared in example 1 with E.coli in different concentrations
Taking 100 mu L of escherichia coli to shake culture in 20mL liquid culture medium at 37 ℃ in a shaking table until the bacterial concentration OD is reached6001.0-2.0, dispensing into small EP tubes, balancing, centrifuging at 13000rpm for 2 min, discarding supernatant, washing bacteria with 20mM PBS buffer (pH 7.4) for 2-3 times, resuspending bacteria with PBS buffer, and making OD600Bacterial solutions with values of 2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.025.
Dissolving the probe in DMSO to prepare 2mM mother liquor, adding 1mL of PBS solution of Escherichia coli with different concentrations into 5 μ L of probe respectively, blowing and beating uniformly, wherein the final concentration of the probe is 10 μ M, and detecting fluorescence under 345nm excitation wavelength to obtain fluorescence spectrum figure 8.
In FIG. 8, the peaks at 375nm and 482nm were low only in the presence of the probe, and the peaks at 375nm were slightly higher and 4nm were slightly higher after addition of E.coliThe peak at 82nm is clearly elevated. The peak at 375nm gradually increased with increasing E.coli concentration; while the peak at 482nm tends to increase first and then decrease. In particular, the concentration of Escherichia coli is determined from OD6000.025 to OD600The peak at 482nm increases with increasing E.coli concentration from OD 6001 to OD600The peak at 482nm decreases with increasing escherichia coli concentration, 4.
Example 10
Detection of different bacteria by the fluorescent probes prepared in example 1
Shaking culturing bacteria 100 μ L and 20mL liquid culture medium at 37 deg.C until bacteria concentration OD6001.0-2.0, dispensing into small EP tubes, balancing, centrifuging at 13000rpm for 2 min, discarding supernatant, washing bacteria with 20mM PBS buffer (pH 7.4) for 2-3 times, resuspending bacteria with PBS buffer, and adjusting OD6000.5; the cultivation of various bacteria (Escherichia coli, Pseudomonas aeruginosa, Agrobacterium, Staphylococcus aureus, Bacillus subtilis, enterococcus faecium, Citrobacter freundii, Chromobacterium violaceum, Bacillus cereus, Staphylococcus epidermidis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae) was performed as described above.
Dissolving the probe in DMSO to prepare 2mM mother liquor, adding 1mL of bacterial liquid into 5 μ L of probe, blowing and beating uniformly, wherein the final concentration of the probe is 10 μ M, detecting fluorescence under 345nm excitation wavelength to obtain a fluorescence spectrogram 9a, and repeating the test of the same kind of bacteria for 5 times. Change in fluorescence ratio (I)482/I375) On the abscissa, the fluorescence increases (Δ S/S)0) Two-dimensional plots 9b were generated for the ordinate to distinguish between different species of bacteria.
In FIG. 9a, the peaks at 375nm and 482nm were low in the presence of the probe only, and the peaks at 375nm and 482nm increased significantly after the addition of the bacteria. The fluorescence enhancement degree is different when different bacteria are added, which is caused by the difference of the surface structures of different bacteria.
The two-dimensional fluorescence ratios of the PI-3 probe of FIG. 9b after the action with different bacteria were obtained by converting the fluorescence intensity of FIG. 9aThe abscissa of the rate chart is the fluorescence intensity ratio I of 482nm to 375nm emission peaks after the PI-3 probe acts on various bacteria482/I375The ordinate is the absolute difference Delta S between the integrated fluorescence areas before and after the action of the probe and various bacteria and the integrated fluorescence area S in the presence of only PI-3 probe0The ratio of (a) to (b). It can be seen that the different bacteria are in different positions in the figure and have almost no crossover, and that the 5-fold reproducibility of the same bacteria is better, substantially within a small area marked by an ellipse.

Claims (9)

1. A fluorescent probe is characterized in that the molecular structural formula of the probe is as follows:
Figure FDA0002311001000000011
2. the method for synthesizing the fluorescent probe as claimed in claim 1, which comprises the following steps:
(1) synthesizing an intermediate 3-bromo-1-pyrenyl acetone:
under the protection of nitrogen, dissolving pyrene and 3-bromopropionyl bromide in dichloromethane, cooling to-5-0 ℃ in an ice salt bath, slowly adding anhydrous aluminum chloride into the reaction liquid under stirring, and stirring for 1-3 hours to obtain the reaction liquid with a dark color; heating the reaction temperature to room temperature, continuously stirring for 6-10 hours, pouring the reaction liquid into ice water, extracting with dichloromethane, drying with anhydrous magnesium sulfate, filtering, removing the solvent by spinning, and separating by silica gel column chromatography to obtain 3-bromo-1-pyrenyl acetone;
(2) synthesis of intermediate 1- (3-bromopropyl) -pyrene:
under the protection of nitrogen, dissolving anhydrous aluminum chloride and lithium aluminum hydride in diethyl ether, dissolving 3-bromo-1-pyrenyl acetone in dichloromethane, slowly adding the dichloromethane into the reaction solution, stirring at room temperature for reaction for 0.5-3 hours, pouring the reaction solution into ice water, quenching the rest of aluminum chloride and lithium aluminum hydride, adjusting the pH of the reaction solution to acidity by hydrochloric acid, then extracting by dichloromethane, drying by anhydrous magnesium sulfate, filtering, removing the solvent by spinning, and performing silica gel column chromatographic separation to obtain 1- (3-bromopropyl) -pyrene;
(3) synthesis of ratiometric fluorescent Probe PI-3 in response to Membrane Charge:
under the protection of nitrogen, dissolving biimidazole and 1- (3-chloropropyl) -pyrene in acetonitrile, heating to 80-140 ℃, reacting for 24-96 hours under stirring, stopping the reaction, separating out white precipitate, filtering, and washing the precipitate with diethyl ether; the obtained precipitate was then dissolved in methanol, to which a saturated methanol solution of potassium hexafluorophosphate was slowly added dropwise to precipitate a white precipitate product, i.e., ratiometric fluorescent probe PI-3, which responded to membrane charge.
3. The method for synthesizing a pyrene-derived fluorescent probe according to claim 2, wherein in the step (1), pyrene: the mass ratio of the 3-bromopropionyl bromide is 1: 0.3-2;
pyrene: the mass ratio of the anhydrous aluminum chloride is 1: 0.3-3;
the volume ratio of the mass of pyrene to the volume of dichloromethane is 1:5-50 g/mL.
4. The method for synthesizing a pyrene-derived fluorescent probe according to claim 2, wherein in the step (2), the intermediate 3-bromo-1-pyrenylacetone: the mass ratio of the lithium aluminum hydride is 1: 0.2-1;
intermediate 3-bromo-1-pyrenyl acetone: the mass ratio of the anhydrous aluminum chloride is 1: 0.5-2;
the volume ratio of the mass of the intermediate 3-bromo-1-pyrenyl acetone to the diethyl ether is 1:3-15 g/mL.
The volume ratio of the mass of the intermediate 3-bromo-1-pyrenyl acetone to the dichloromethane is 1:15-90 g/mL.
5. The method for synthesizing a pyrene-derived fluorescent probe according to claim 2, wherein in the step (3), the intermediate 1- (3-bromopropyl) -pyrene: the mass ratio of the bigeminal imidazole is 15-4: 1;
the volume ratio of the mass of the intermediate 1- (3-bromopropyl) -pyrene to the acetonitrile is 1:20-200 g/mL.
6. A fluorescence method for rapidly detecting bacteria based on the fluorescent probe of claim 1 is characterized in that the method specifically comprises the following steps:
1) shaking and culturing the bacteria at 37 ℃ by a shaking table;
2) the bacteria were centrifuged at 13000rpm for 2 minutes, the supernatant was discarded, the bacteria were washed 2-3 times with 20mM PBS buffer at pH 7.4, and finally the bacteria were resuspended in PBS buffer;
3) the bacteria were probed with a fluorescence spectrometer.
7. The fluorescence method for rapidly detecting bacteria according to claim 6, wherein: culturing the bacteria to OD in step 1)600＝0.1-2.0。
8. The fluorescence method for rapidly detecting bacteria according to claim 6, wherein: step 2) resuspension to OD of bacteria with PBS6000.1-2.0, final concentration of probe 5-20 μ M.
9. The fluorescence method for rapidly detecting bacteria according to claim 6, wherein: step 3), detecting fluorescence at 345nm excitation wavelength to obtain fluorescence spectrogram, and changing the fluorescence ratio by fluorescence enhancement (I)482/I375) As probe response signals for distinguishing different species of bacteria.
CN201911258659.6A 2019-12-10 2019-12-10 Fluorescence method for rapidly detecting bacteria based on fluorescent probe Pending CN112940716A (en)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN110511186A (en) * 2018-05-22 2019-11-29 中国科学院大连化学物理研究所 The ratio fluorescent probe that a kind of pair of membrane charge responds and its application in Bacteria Detection

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110511186A (en) * 2018-05-22 2019-11-29 中国科学院大连化学物理研究所 The ratio fluorescent probe that a kind of pair of membrane charge responds and its application in Bacteria Detection

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
SHUANGSHUANG LONG等: ""A self-assembly/disassembly two-photo ratiometric fluorogenic probe for bacteria imaging"", 《CHINESE CHEMICAL LETTERS》 *
SHUANGSHUANG LONG等: ""Rapid Identification of Bacteria by Membrane-Responsive Aggregation of a Pyrene Derivative"", 《ACS SENS.》 *
SHUANGSHUANG LONG等: ""Self-assembling nanoprobes that display two-dimensional fluorescent signals for identification of surfactants and bacteria"", 《CHEM. COMMUN.》 *

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